The newly-discovered adipocyte hormone leptin is hypothesized to provide information about the size of fat stores to central receptors regulating energy balance. The signal(s) that up regulate leptin expression with obesity are know known. the goal of this application is to elucidate the cellular mechanisms linking increased fat stores to increased leptin expression. Evidence from the few humans studies conducted to date demonstrate that in addition to percent body fat, chronic nutritional state (e.g. hypoccaloric feeding, prolonged hyperisulinemia) but not individual meals, appear to influence leptin expression. In contrast, leptin expression in rodent adipose tissue is acutely responsive to feeding. Thus, studies of leptin regulation in human adipose tissue, and in particular studies of its long-term regulation, are needed. Preliminary data from in vivo and in vitro experiments show that insulin and glucocrticioid effects o leptin expression are dependent on the degree of obesity. Additionally, because differences in leptin regulation between omental and subcutaneous adipose tissue have ben observe in preliminary experiments, both depots will be studied. Adipose tissue samples will be obtained from lean, moderately obese and severely obest subjects and placed in organ culture with varying concentrations of insulin and/or glucocorticoid for up to 7 days. The cellular mechanisms underlying insulin and glucocorticoid effects on leptin mRNA levels and the secretion of immunoreative leptin protein will be established. We will specifically investigate the hypothesis that the insulin insensitivity that accompanies fat cell enlargement is an important determinant of increased leptin expression in obesity. to determine whether leptin production is also regulated at post-transcriptional levels, pulse- chase studies will examine rates of leptin biosynthesis and secretion. The proposed studies will provide basic knowledge of how leptin expression is regulated in human adipose tissues in relationship to fat cell size. This information is central to understanding the role of this hormone system in the pathogenesis of human obesity.